Agouti-related protein (AGRP) and agouti (ASP) are the only two naturally occurring antagonists of G protein coupled receptors (GPCRs) identified to date, and when over expressed in mice, result in an obese phenotype. Obesity (body mass index >25) afflicts millions of people in the United States (an estimated 97 million adults in the U.S.) and other countries, and is a major risk factor for heart disease, type II diabetes mellitus, stroke and hypertension. In industrialized countries, the problem of obesity is compounded by overeating, a high fat content diet, and a lack of exercise. The last few years have seen the characterization of over 25 pathways that have been identified to participate in and regulate feeding behavior and energy homeostasis. The melanocortin pathway includes five such genetic factors that have been demonstrated to mediate weight homeostasis, and when modified, result in obesity. The melanocortin pathway includes the melanocortin agonists, derived from the preprohormone proopiomelanocortin (POMC) gene transcript, the five melanocortin receptors identified to date (MC1R-MC5R), and the only 2 naturally occurring antagonists of GPCRs, agouti (ASP) and agouti-related protein (AGRP). The five melanocortin genetic factors identified as being involved in energy homeostasis are POMC, ASP, AGRP, and the brain specific melanocortin-4 (MC4R) and the melanocortin-3 (MC3R) receptors. AGRP and ASP are paracrine factors that antagonize the melanocortin receptors. Specifically, ASP antagonizes the skin melanocortin-1 receptor (MC1R) and the brain MC4R. AGRP normally only antagonizes the melanocortin receptors in the brain, MC3R and MC4R. A polymorphism has been identified in humans diagnosed with anorexia nervosa, linking AGRP with a human physiological disease state. Both ASP and AGRP are 131-132 amino acids in length (depending upon species), and possess a C-terminal domain that putatively contains 5 disulfide bridges. The C-terminal domain has been identified as the region of the protein that possesses antagonistic properties at the melanocortin receptors. These data support the hypothesis that AGRP and ASP are involved in the regulation of melanocortin receptor mediated physiological pathways. The research objectives of this proposal are to 1) identify structural determinants of the AGRP protein important for molecular recognition and functional activity at melanocortin receptor proteins, and 2) identify putative AGRP-MC4 receptor interactions. In addition to generating structural and functional information about one of the only two endogenous GPCR antagonists, new tools will be developed which may be utilized in vivo to gain a broader understanding of the physiological mechanism(s) resulting in obesity and skin pigmentation. [unreadable] [unreadable] [unreadable]